Lachnellula species that appear to have lost the need for the primary MAT1-1-1 protein. Ancestral state reconstruction enabled the identification of numerous transitions between homothallism and heterothallism in the Leotiomycetes and suggests a heterothallic ancestor for this group. This comprehensive catalog of mating-related genes from such a large group of fungi provides a rich resource from which in-depth, functional studies can be conducted in these economically and ecologically important species.The Leotiomycetes is a hugely diverse group of fungi, accommodating a wide variety of important plant and animal pathogens, ericoid mycorrhizal fungi, as well as producers of antibiotics. Despite their importance, the genetics of these fungi remain relatively understudied, particularly as they don’t include model taxa. For example, sexual reproduction and the genetic mechanisms that underly this process are poorly understood in the Leotiomycetes. We exploited publicly available genomic and transcriptomic resources to identify genes of the mating-type locus and pheromone response pathway in an effort to characterize the mating strategies and behaviors of 124 Leotiomycete species. Our analyses identified a putative a-factor mating pheromone in these species. This significant finding represents the first identification of this gene in Pezizomycotina species outside of the Sordariomycetes. A unique mating strategy was also discovered in  Neurospora crassa , two sen mating , 28. Thet manner , 29, 30.Saccharomyces cerevisiae, with a simple peptide pheromone expressed by the α-cell and a lipopeptide pheromone being expressed by the a-cell [N. crassa [Cryphonectria parasitica [Magnaporthe grisea [Huntiella omanensis [F. graminearum [Sordaria macrospora [MAT locus. The α-factor pheromone has also been identified from species in classes outside of the Sordariomycetes, including the Eurotiomycetes [Genes encoding the fungal mating pheromones were first identified in e a-cell , 32. Thi. crassa , Cryphonrasitica , Magnapoe grisea , and Hunmanensis  harbor bminearum  and Sordcrospora , but theomycetes , 37 and omycetes . In contBotrytis cinerea [Rhynchosporium secalis [Oidium neolycopersici [Hymenoscyphus fraxineus [Hyaloscypha species [Gamarada debralockiae [Pseudogymnoascus destructans [Ascocoryne sarcoides, [Glarea lozoyensis, [Erysiphe pisi [B. cinerea [The Leotiomycetes is an incredibly diverse class of fungi . It incl cinerea , Rhyncho secalis , Oidium opersici  and Hymeraxineus  as well  species  and Gamaalockiae . The grotructans , and thorcoides, ) and antoyensis, ). Some ophe pisi  and thos cinerea . Yet, th cinerea .The many important impacts of meiosis on the life cycles of fungi demand a greater number of studies to fully understand the capacity of these fungi to sexually reproduce. Undergoing meiosis allows for the production of genetic diversity as well as the purging of deleterious alleles, which may allow for an increased rate of adaptation to changing environmental conditions . AdditioThe motivation for this study lay in the recognition that the Leotiomycetes do not include model species that have been utilized to elucidate the genetics underpinning sexual reproduction and that little is known regarding this process in these fungi. We thus used comparative genomics tools and mined 124 publicly available genomes for genes encoding the mating-type proteins as well as the mating pheromones and their cognate receptors. The emerging information was then used to trace transitions between heterothallism and homothallism in the class. This is the first study, to our knowledge, to systematically catalog and compare these genes in the Leotiomycetes and it provides a rich resource from which in-depth, functional studies can be conducted in these non-model fungi.incertae sedis species whose taxonomic placement within the Leotiomycetes was not clear  in December 2021. Of these, nine were excluded from further analysis due to their poor assembly statistics . A furthot clear . Additioot clear .B. cinerea gene models were used for gene prediction.Many of the genomes that were used had associated GFF files that were also downloaded  and usedNumerous genes involved in mating (as detailed below) were found to have incorrect gene models in the downstream applications. This was true of the annotations downloaded from NCBI as well as the annotations predicted by AUGUSTUS. It was possible to identify these errors because the encoded proteins did not harbor the correct functional domains, or the genes possessed an unexpected number of introns. Additionally, some genes, particularly those encoding the two mating pheromones were not annotated at all. In these cases, we produced alignments using homologs from close relatives to determine whether an appropriate model could be manually produced. Additionally, when available, we mapped RNAseq reads to confirm intron-exon boundaries.Create Alignment function in the Alignments and Trees module, with default settings. The RNA-based mappings, using trimmed RNA-seq reads, were produced using the Map Reads to Contigs function in the De Novo Sequencing module with default settings, a minimum similarity fraction of 0.8, and a minimum length fraction of 0.5. The alignments and mappings were manually inspected, curated, and used for the annotation of the gene regions in question.DNA- and RNA-based alignments and mappings were produced in CLC Main Workbench V22.0 and CLC Genomics Workbench V22.0, respectively. The DNA-based alignments, using the gene sequences from related species, were generated using the Throughout this paper, we have described genes that were not present and/or intact as deleted, missing, truncated, fragmented, or that they could not be annotated. The terms deleted and missing referred to genes which could not be found within the genome at all, even when using tBLASTn and synteny-based analyses. These genes have been lost from the genome entirely. Genes that were described as truncated or fragmented were genes whose 5’ or 3’ regions could be identified using BLAST analyses but that did not encode for the full-length proteins. Lastly, genes that could not be annotated typically referred to genes where partial regions could be identified using BLAST but there had been sufficient degeneration or pseudogenization of the gene so that no suitable gene and CDS annotations could be made.MAT genes are commonly associated with Leotiomycete species; MAT1-1-1, MAT1-1-3, MAT1-1-5, MAT1-2-1 and MAT1-2-10 . VariouAT locus . These tMAT loci was used to predict the sexual strategy of each species. Species were labelled as heterothallic if genes from only a single idiomorph were present in the representative genome. In contrast, when genes from both idiomorphs were present, the species were labelled as homothallic or undetermined. Species were classified as homothallic when genes from both idiomorphs were identified within a single locus. Many of the species that were categorized as undetermined typically harbored truncated MAT1-1-1 or MAT1-2-1 genes, making it difficult to predict whether they were capable of sexual reproduction at all. Alternatively, these undetermined species possessed genes from both idiomorphs, but that were assembled into different loci.The gene content of the MAT1-1-1 gene flanking the MAT1-2-associated genes.While the majority of homothallic species harbored genes suggestive of primary homothallism, a few species exhibited evidence for mating type switching, a form of secondary homothallism. In these species, it was possible to identify the genetic signals of switching, including the presence of identical repeats and/or fragments of the The α-factor pheromone is typically the easiest of the two mating pheromones to identify as it encodes a protein with a recognizable and unique structure. The full length, pro-pheromone protein harbors a signal peptide at the N-terminal and thus begins with a hydrophobic region. This is followed by repeats of a conserved 9–14 amino acid sequence representing the mature pheromone. These conserved repeats are typically preceded by an STE13 recognition sequence  and followed by the KEX1/2 recognition sites .S. sclerotiorum and B. cinerea ppg1 gene and protein sequences  and only has a single strictly conserved character- a C-terminal CAAX or CPAX domain . This domain is not exclusive to the a-factor pheromone and thus the identification of this gene requires additional evidence from other data.Methods similar to those used to identify the α-factor pheromone were used in an attempt to identify the a-factor pheromone. BLAST-dependent analyses were conducted using the a-factor pheromones from more than 40 Sordariomycetes and yeasts  38, 60––60–62. MB. cinerea, B. sinoalli, S. sclerotiorum, M. fructicola, R. commune, A. resinae, and P. destructans) were extracted and translated into predicted protein sequences. These lists were manually filtered to retain only proteins ≤ 100 aa and harboring a terminal CAAX or CPAX domain  and ste3 (a-factor receptor), were identified using BLASTn and tBLASTn analyses, using B. cinerea and S. sclerotiorum gene and protein sequences  and the NCBI Conserved Domain Search . In some cases, the functional domains were not detectable and/or an incorrect number of transmembrane domains (i.e.: ≠7) were annotated. These genes were subjected to manual re-annotation as described above, using DNA and RNA alignment in an effort to produce proteins with the correct domains.The two pheromone receptor genes, equences . Once idleotiomycetes_odb10 database was used. Orthologs that were common to all taxa studied were included in the phylogenomic analyses below. Amino acid sequences were aligned within each data file with MAFFT v. 7.407 [The phylogenomic analyses were conducted in a manner comparable to other recent studies using large genomic datasets from diverse fungi –65. Singv. 7.407 , 69 usinv. 7.407  and dataA. pellizariae as the outgroup taxon. A supertree was generated from all gene trees obtained above. This was done using ASTRAL version 5.7.8 [Phylogenetic analyses were conducted on a concatenated dataset as well as on individual gene sequences. FASconCAT-G v1.0 was used to generate a concatenated dataset (a supermatrix) as well as a file defining the partitions. Maximum likelihood analyses were performed with IQTree v.2.2 , 72 to con 5.7.8  with defAncestral state reconstruction of mating systems was conducted to assess their evolution over the phylogeny of the species considered. A Bayesian approach was followed by employing MrBayes Ancestral States with R (MBASR) . The phyape and phytools packages, and the plotTree function [The phylogeny presented in function , 79. ChaOf the 3 234 BUSCO  genes in the Leotiomycete database, 76 were present as complete, single copy orthologs in all 124 genomes included in this study. Of these, 72 gene alignments possessed more than 200 characters and were subsequently used for the phylogenomic analyses. The phylogenies produced using the concatenated dataset and the individual gene trees had similar topologies with minor variation . The topMAT loci were identified and annotated from 104 of genomes considered in this study. Most of these genomes harbored genes that have been previously described from the MAT loci of the Leotiomycetes, including MAT1-1-1, MAT1-1-3, MAT1-1-5, MAT1-1-2 and MAT1-2-10 .Intact AT1-2-10 . In addiMAT1-1-13 following the Pezizomycotina MAT nomenclature system [The novel gene is short, only 330 bp, and is made up of a CDS with two exons interrupted by an intron and encodes a protein of only 90 aa . No homoe system .Sexual reproduction in the Leotiomycetes was found to include heterothallism, primary homothallism and mating type switching . Genes fMAT1-1 and MAT1-2 idiomorphs were identified. Of these, 10 had MAT locus structures that were suggestive of primary homothallism while a further five harbored MAT loci that were indicative of mating type switching [MAT1-1 and MAT1-2-associated genes, it was not possible to predict the sexual strategy of these isolates  were present within the two MAT1-1-1 fragments and would likely facilitate an inversion event, allowing for the generation of an intact MAT1-1-1 gene.The rotiorum . The isoMAT loci of Sclerotinia glacialis and Sclerotinia borealis exhibited unique gene structures, which may be indicative of mating type switching or heterothallism . Two of these, Phialocephala sp. D728 and Phialocephala subalpina, had MAT loci characteristic of heterothallic fungi, possessing the MAT1-1 (MAT1-1-1 and MAT1-1-3) and MAT1-2 (MAT1-2-1) idiomorphs, respectively. These MAT loci were also flanked by SLA2 and APN2. However, a third species, Phialocephala scopiformis, harbored the MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes, which, in contrast, were associated with a SAM-Mtase gene [P. scopiformis is homothallic and suggested a transition towards homothallism in this genus as well as in the family.There were three genomes available from the genus ase gene . This inHymenotorrendiella dingleyae, as the MAT1-1-associated genes were flanked by APN2 and SLA2 while the MAT1-2-1 gene was present elsewhere in the genome. This might be indicative of homothallism, but could also mean that the genome was generated from a mixed culture of MAT1-1 and MAT1-2 individual(s). Additionally, it was impossible to determine the sexual strategy of Hymenoscyphus linearis and Hymenoscyphus herbarum. The genome of H. linearis encodes an intact MAT1-1-1 gene but both the MAT1-1-3 and MAT1-2-1 genes were truncated. In contrast, H. herbarum contains an intact MAT1-1-3 and MAT1-2-1 genes, but no MAT1-1-1 gene. It is thus unclear whether these species are capable of sexual reproduction- given the absence of typically essential mating type genes.In total, 14 Helotiaceae species were used in this study, five of which were confirmed to be heterothallic and six of which were thought to be homothallic . It was MAT1-1-1 and MAT1-1-3 [e.g. H. fraxineus, [MAT1-1-1, MAT1-1-3 and MAT1-1-5  to 446 aa with 18 mature repeats (Botrytis fragariae). Additionally, the vast majority of mature repeats had either a GQ or PG dipeptide, a sequence that facilitates the β-turn necessary for the functioning of this pheromone in F. oxysporum and S. cerevisiae, respectively [Putative α-factor pheromone genes were identified in 115 of the 124 species included in this study  Files. Oectively .Hymenoscyphus repandus, had a signal peptide and recognizable KEX1/2 and STE13 processing sites but the mature pheromone peptide sequences were difficult to distinguish. There were two GQ dipeptides that were used to identify the putative mature peptides, but in both cases, the processing sites weren’t proximal to the repeats. Additionally, Hyphodiscus hymeniophilus exhibited characteristics associated with the α-factor pheromone, however, fewer KEX1/2 processing sites were present than expected. Instead, putative STE13 sites were found after numerous repeats, suggesting that this species might rely more heavily on STE13-mediated processing.Two species harbored α-factor pheromone genes which, while recognizable, were divergent in terms of sequence and/or structure . One of Blumeriella jaapii, encoded for a putative protein that was structurally different to the other α-factor pheromones in that it did not harbor multiple repeats of a mature pheromone peptide or recognizable KEX1/2 and STE13 processing sites. However, each of the nine homologs encoded proteins harboring a signal peptide, a single potential mature peptide containing a GQ dipeptide, and a putative STE13 processing site . It is unclear whether this protein represents a pre-protein that requires further processing, analogous to the typical α-factor pheromone, or if this is the mature pheromone factor. Neither a typical nor an atypical α-factor could be identified from the other five Erysiphaceae species.Nine species accommodated in the Erysiphaceae possessed divergent α-factor pheromones . This diB. cinerea (4 genes), Botrytis sinoalli (5 genes), S. sclerotiorum (11 genes), Monilinia fructicola (12 genes), Rhynchosporium commune (5 genes), Amorphotheca resinae (2 genes), and P. destructans (2 genes). These genes each encoded small proteins (≤100 amino acids) that terminated in a CAAX or CPAX domain  to 64 (Rhynchosporium agropyri) amino acids in length and most frequently terminated in a CTVM domain, although CVVM, CIVM, CTIL, CSIM, CSVM were also present in a few species  had a number of repeats in common, with RCGHPGQPCG and RCGYPGQPCG occurring in seven and five species, respectively. Lachnaceae species (n = 8) also possessed many repeats in common with one another, with seven harboring the RCGARGQGCW repeat.There was low inter-species, intra-family diversity for the α-factor pheromone. In many of the families considered here, species harbored identical copies of the mature repeat of the α-factor pheromone. In the Sclerotiniaceae (n = 36), 31 species possessed the mature repeat WCGRPGQPC and only nine of the species had additional unique repeats within the same pro-pheromone . SimilarChlorencoelia torta (Cenangiaceae), Venustampulla echinocandica (Pleuroascaceae), and A. pellizariae (Thelebolaceae).In contrast to the conservation seen in most other families, Hyaloscyphaceae species (n = 5), tended to possess repeats unique to each species. This was typically due to the first and last amino acids of the repeats, which were highly variable. Additionally, in families that were represented by a single species, the species frequently possessed unique α-factor pheromone repeats. Examples include Botrytis species and four of the six Monilinia species possessing identical a-factor pheromones , regardless of mating type and tissue type (M. fructicola putative a-factor was undetectable (RPKM = 0.00). Lastly, the MAT1-1 isolate of A. resinae expressed the α-factor at a very low level (RPKM < 1.00) while the putative a-factor was more highly expressed (RPKM = 19.58).In sue type . The expMAT genes were also expressed at very low levels in all three species  and a-factor receptor (ste3) were identified in the majority of the species included in this study. Furthermore, most of these genes encoded proteins with the expected seven transmembrane domains as well as the STE2 (pfam02116) and STE3 (pfam02076) functional domains. There were a number of exceptions that are worth noting, where the gene encoding either of the two receptors has been disrupted and the putative protein is not predicted to contain the expected domains or has been entirely deleted from the genome.Genes encoding both the α-factor receptor  are known to occur [MAT idiomorphs each harbor an allelic HMG protein. Furthermore, HMG proteins are known to be important transcription factors, with a network of HMG proteins controlling the sexual cycle in Sordariomycete species such as P. anserina [Lachnellula species. This would lead to a dependence on HMG proteins and the subsequent loss of the MAT1-1-1 α-box protein. Unfortunately, there is a lack of research specifically focusing on sexual reproduction in Lachnellula species and additional mating studies need to be conducted to determine whether these partners are indeed compatible.One of the most surprising results of this study was the identification of a novel mating strategy in species of to occur . While tto occur , 97. In anserina . Given tWe were able to identify a putative a-factor pheromone in the Leotiomycetes; the first time this class of pheromone has been identified from non-Sordariomycete Pezizomycotina. The presence of other genes from the pheromone response pathway and widespread heterothallism in the Leotiomycetes suggested that the a-factor pheromone would also be present. However, while efforts have been made to identify the a-factor pheromone in other non-Sordariomycete classes, it has not previously been discovered , 99. UsiThe function of the gene that has been identified as the putative a-factor pheromone in the Leotiomycetes is unclear. The fact that components of the pheromone response pathway, including the α-factor pheromone and both pheromone receptors (STE2 and STE3), are conserved across the Ascomycota, and specifically within the Leotiomycetes, suggests that the putative a-factor pheromone is involved in mating. This would be in a manner comparable to the role of the a-factor pheromone in the Sordariomycetes. However, there are a number of lines of evidence that strongly suggest that this is not the case.S. sclerotiorum show that the expression of the α-factor gene is significantly altered in ΔMAT1-1-1, ΔMAT1-1-5, ΔMAT1-2-1, and ΔMAT1-2-10 mutants, further suggesting a mating type-independent control of this pheromone [The most notable line of evidence suggesting that the identified a-factor gene is not involved in mating in the Leotiomycetes, is the fact that the two pheromones were not expressed in a mating type-dependent manner in the three distantly related heterothallic species investigated here. While these three species may not be representative of all the Leotiomycetes, this does indicate that, at least in certain species, these proteins do not act in the same manner as their Sordariomycete homologs, which are expressed strictly according to mating type to facilitate compatible mating partner interactions. Furthermore, functional characterization studies in heromone . In the Fusarium fujikuroi species complex (previously the Gibberella fujikuroi species complex) [The high level of intra-family conservation of the α-factor pheromone as well as the high level of conservation of the a-factor in the Sclerotiniaceae and Rutstroemiaceae also provides evidence against the utility of these proteins as mating pheromones in the classical sense. This contradicts the hypothesis that the pheromones act as signaling molecules that aim to specifically attract opposite mating partners of the same species . The faccomplex) . While tN. crassa, the a-factor pheromone is thought to act as a conglutination factor that cements hyphae together during the production of the ascoma [N. crassa mutants exhibit defects associated with vegetative growth, suggesting that the pheromone may also play an unknown role in filamentous differentiation [Fusarium oxysporum also utilizes components of the pheromone response pathway for a variety of non-mating functions [The widespread occurrence and conservation of the two pheromones throughout the Leotiomycetes, implies that they play important roles in the lifecycles of these species. In other fungi, pheromones and their receptors have been co-opted for new functions, both related and unrelated to mating. For example, in e ascoma . Additiontiation . More reunctions , 104. Thin silico-based predictions and may not be fully accurate. However, to overcome this limitation, the annotations from seven genomes were used to create as comprehensive a list as possible from which to filter out non-target proteins. Secondly, short genes, such as pheromones, are often not annotated during standard annotation pipelines. Thus, while genes encoding proteins as short as 35 aa were annotated in, for example, the S. sclerotiorum genome, it is possible that shorter proteins may have been missed prior to filtering. A BLAST- and annotation-independent method is currently being developed and aims to solve these limitations for future studies.There are limitations to the method used in this study to identify this putative pheromone factor. Firstly, our BLAST-independent method relied on the availability of well-annotated genomes, because the predicted protein lists that were filtered to identify the putative a-factor pheromone proteins were produced directly from the CDS annotations. These annotations are the result of in silico for these structural domains. Thus, while currently available genome annotation pipelines are useful for the identification of the genomic location of a particular gene, it is often necessary to manually curate the annotations, using well-characterized gene sequences from closely related species or transcriptomic data.The identification of numerous, complex genes across the Leotiomycetes illustrated the lack of high-quality gene models in many publicly available genomes. This was particularly true in species for which limited non-genomic NGS data are available (i.e.: RNA-seq data) and was specifically evident when the genes encoding the two pheromone receptors were considered. Both receptors typically have numerous intron/exon boundaries and produce proteins with a very strict structure/function relationship. The structure of these proteins contributes directly to their functioning. The extracellular N-terminal, transmembrane domains, and intracellular C-terminal are essential for signal recognition, anchoring of the receptors, and transmitting the intracellular signal, respectively. This strong relationship made identifying misannotated genes possible as each encoded protein could be assessed The Leotiomycetes accommodates an incredibly diverse group of fungi, including well-known plant and animal pathogens as well as species important for various biotechnological applications. Sexual reproduction in this group of fungi is poorly understood. However, recent advances in sequencing technologies made it possible in this study to compare the genomes of 124 Leotiomycete species to determine whether key genes involved in sexual reproduction were present and conserved. This led to the identification of a putative a-factor pheromone, the discovery of a unique mating strategy, and evidence supporting heterothallism as the ancestral mating strategy of these fungi. Other than these important new insights, this study provides a rich database from which future functional studies can be conducted on these ecologically and economically relevant fungi.S1 Fig(PPTX)Click here for additional data file.S2 Fig(PPTX)Click here for additional data file.S3 Fig(PPTX)Click here for additional data file.S4 Fig(PPTX)Click here for additional data file.S5 Fig(PPTX)Click here for additional data file.S6 Fig(PPTX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.S9 File(PDF)Click here for additional data file.S10 File(PDF)Click here for additional data file.


To explore the prognostic significance of PET/CT-based radiomics signatures and clinical features for local recurrence-free survival (LRFS) in nasopharyngeal carcinoma (NPC). We retrospectively reviewed 726 patients who underwent pretreatment PET/CT at our center. Least absolute shrinkage and selection operator (LASSO) regression and the Cox proportional hazards model were applied to construct Rad-score, which represented the radiomics features of PET-CT images. Univariate and multivariate analyses were used to establish a nomogram model. The concordance index (C-index) and calibration curve were used to evaluate the predictive accuracy and discriminative ability. Receiver operating characteristic analysis was performed to stratify the local recurrence risk of patients. The nomogram was validated by evaluating its discrimination ability and calibration in the validation cohort. A total of eight features were selected to construct Rad-score. A radiomics–clinical nomogram was built after the selection of univariate and multivariable Cox regression analyses, including the Rad-score and maximum standardized uptake value (SUVmax). The C-index was 0.71 (0.67–0.74) in the training cohort and 0.70 (0.64–0.76) in the validation cohort. The nomogram also performed far better than the 8th T-staging system with an area under the receiver operating characteristic curve (AUC) of 0.75 vs. 0.60 for 2 years and 0.71 vs. 0.60 for 3 years. The calibration curves show that the nomogram indicated accurate predictions. Decision curve analysis (DCA) revealed significantly better net benefits with this nomogram model. The log-rank test results revealed a distinct difference in prognosis between the two risk groups. The PET/CT-based radiomics nomogram showed good performance in predicting LRFS and showed potential to identify patients at high-risk of developing NPC. Radiotherapy, with some cases requiring the addition of chemotherapy, is the standard treatment for NPC. Intensity-modulated radiation (IMRT) is a revolutionary radiotherapy technique that has considerably improved the treatment outcomes of patients with NPC, particularly in terms of local control. Although extensive use of IMRT has contributed to excellent local control, local recurrence remains an important cause of treatment failure in ~ 10% of advanced NPC cases. Owing to the potential existence of a disease with intrinsic radioresistance, the salvage treatment options are complex and limited for patients with locally recurrent NPC and typically present with a poor prognosis for patients with rT3–T4 disease5. Local recurrence plays a particularly vital role in treatment failure. Therefore, a more accurate prediction method is required to identify the individuals who are resistant to treatment and susceptible to recurrence and metastasis. Adjusting the prediction-based therapeutic strategy could provide a more favorable outcome. Although the American Joint Committee on Cancer TNM (AJCC/TNM) staging system is the key to prognosis prediction and risk stratification, the prediction of local failure still can be improved in some ways6.Nasopharyngeal carcinoma (NPC) is a rare cancer with a highly asymmetric geographical distribution. According to the International Agency for Research on Cancer, approximately 129,000 new cases of NPC were diagnosed worldwide in 2018. The majority of new cases were reported in East Asia and Southeast Asia7. The spectrum of information provided by 18F-FDG PET/CT improves the early detection of cancer and consequently the treatment outcome. Coincidentally, it has excellent utility in combination with radiomics. Radiomics, a relatively new field aimed at connecting medical imaging information with biological and clinical endpoints, has grown into a promising field in oncology. By translating medical imaging into mineable, high-dimensional, and quantitative imaging features via high-throughput extraction of data-characterization algorithms, radiomics provides a brand new and dependable way to strengthen the predictive ability of a prediction model10. To our knowledge, the predictive capability of 18F-FDG PET/CT features for local failure NPC remains unclear. Thus, this research aimed to establish a radiomics signature based on 18F-FDG PET/CT to predict local failure.18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) imaging is becoming widely implemented because of its better sensitivity and specificity than conventional anatomical imaging techniques like CTThis retrospective study enrolled 726 patients with newly diagnosed NPC who underwent complete treatment at our center between January 2012 and December 2018. We included patients (a) with a biopsy-confirmed primary NPC; (b) with comprehensive baseline clinical data available; (c) with pretreatment whole-body 18F-FDG PET/CT findings available; and (d) having undergone radical treatment. We excluded patients with (a) distant metastasis; (b) other malignancies; (c) a concomitant fatal disease; (d) a history of cancer treatment; and (e) insufficient follow-up data available. All patients were staged according to the 8th edition of the AJCC/TNM staging system. Our study was performed in accordance with the Helsinki Declaration. The Ethics Committee of the Fujian Cancer Hospital authorized this study (YKT2020-011-01).The whole-body [18F] FDG PET/CT scan before treatment was performed using a Gemini TF-64 PET/CT scanner , and 18F-FDG was manufactured by HM-10 cyclotron. Patients fasted for over 6 h to maintain a serum blood glucose level of 3.9–6.5 mmol/L before radiotracer injection. Then, images were acquired 40–60 min after FDG injection at a dose of 148–296 MBq. A low-dose CT from the head to the proximal thigh  was obtained for attenuation correction and fusion purposes. PET image reconstructions were finished using the manufacturer’s proprietary Blob-ordered-subset-time-of-flight algorithm.http://www.manteiatech.com/). The primary tumor was delineated on PET-CT imaging by a single observer  under the guidance of an experienced superordinate physician (20 years of experience). Pretreatment enhanced MRI and/or CT images were used as a guide for PET-CT delineation. The ROI of the primary tumor (ROI-P) was semi-automatically segmented based on the region with an SUVmax of more than 2.5. Each ROI was evaluated and manually adjusted, if necessary. The ROI-P of CT was automatically generated from the corresponding PET volumes.The entire segmentation process was performed using Accucontour software , wavelet, sigma, square, square root, logarithm, gradient, and exponential], which were obtained from ROI-P in both PET and CT images using the Pyradiomics package (version 2.12). The fixed bin width was set to 0.1 g/mL for PET images, 25 HU for CT images. The features could be categorized as follows: shape-based features, first-order statistics, and texture features .P < 0.05). Then, Spearman’s correlation analysis was performed to reduce the redundancy of radiomics features. One of the paired significantly correlated features was removed by completely random .To increase algorithmic stability and performance, all features were scaled using Z-score normalization. Next, the univariate analysis was first used to select the relevant radiomics features with LRFS  regression and the Cox proportional hazards model for predicting local recurrence-free survival (LRFS) of patients with NPC in the training set. The training cohort was split as follows: 80% of the data represented the verification cohort for parameter selection and model construction and 20% of the data represented the test cohort for model validation. Three-fold cross-validation was performed, and the Rad-score and clinical parameters were subjected to univariate analysis. Then, statistically significant variables were included in multivariable Cox regression analysis to identify independent prognostic factors. Then, the factors identified were used to establish a radiomics–clinical model. A nomogram was constructed to predict 2- and 3-year LRFS rates on the basis of the radiomics–clinical model. The concordance index (C-index) and area under the receiver operating characteristic curve (AUC) analysis were used to evaluate the nomogram's discriminating power. The calibration curve was derived to compare the observed and predicted probabilities. DCA was used to evaluate the clinical validity of the nomogram by quantifying the net benefits at different threshold probabilities. Then, the ROC curve was used to classify the patients into two risk groups. Using the Kaplan–Meier method of survival analysis, survival curves for the different risk groups were plotted and then compared using the log-rank test.11. The prescribed radiotherapy doses were as follows—GTV: 70–72.6 Gy/31–33 fx, CTV1: 62–62.7 Gy/31–33 fx, and CTV2: 54.4–56.2 Gy/31–33 fx.Platinum-based chemotherapy was delivered to 682 patients . In the entire cohort, 624 (86%), 532 (73.3%), and 162 (22.3%) cases underwent neoadjuvant, concurrent, and adjuvant chemotherapy, respectively. The most commonly used regimen was platinum + paclitaxel/gemcitabine. Intensity-modulated radiotherapy (IMRT) was used for radiotherapy. The target volume and radiotherapy dose were implemented according to a previously described protocolNotably, LRFS, which was defined as the time from pathological diagnosis to local relapse or the end of follow-up, was selected as the primary endpoint. All patients had a regular follow-up schedule—every 3 months for 2 years, every 6 months for years 3–5, and then annual visits.P value of < 0.05.All analysis were performed using R and Python. The Chi-squared test was used to validate differences between the two variables. The ROC curve determined the optimal cutoff value for Rad-score, EBV-DNA, SUVmax, and other continuous variables to predict local recurrence with the maximum value of the Youden’s index. The log-rank test was used to assess the statistical significance of differences between the two survival groups in Kaplan–Meier survival analysis. A statistically significant difference was indicated by a two-sided The study was approved by the Ethical Committee of Fujian Cancer Hospital (YKT2020-011-01) and was conducted in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Patient identifiers, such as names, were not collected, and instead, patients were given a numerical identifier. Informed consent was obtained from all participants, and for those aged under 18 years, the consent was obtained from a parent or legal guardian. For confidentiality, the patients’ charts were used only within the confines of the records department, and only the investigators and study assistant had access to the files.P range: 0.090–1.000). The median follow-up times for training and validation groups were 48 months (range: 3–118 months) and 50.5 months (range: 9–118 months), respectively. There were 50 cases of local recurrence in the training group and 13 such cases in the validation group. Furthermore, the 2- and 3-year LRFS rates for the training cohort were 95.4% and 93.6%, respectively. The 2- and 3-year LRFS rates for the validation cohort were 97.3% and 93.1%, respectively. The above information indicates that the patients in the training and validation cohorts had balanced distribution of survival and baseline characteristics.A total of 726 patients were recruited for our research. To construct and validate the nomogram, patients were randomly divided into a training cohort (n = 580) and validation cohort n = 146) in an 8:2 ratio. Table 46 in an A total of 85 features remained after dimensionality reduction, which included Spearman’s correlation and univariate analysis. To avoid over-fitting, we used LASSO regression and the Cox proportional hazards model to identify potential features and build the Rad-score model. The α value 0.0108) was determined during cross-validation, which made the C-index of the verification cohort have the highest mean value . Details of LASSO regression and the Cox proportional hazards model are shown in Fig. 08 was deRad-score = 0.23*PET-gradient_glcm_Clustershade + 0.06*PET-logarithm_glcm_ldn-0.55*PET-square_gldm_SmallDependenceEmphasis-0.54*PET-square_ngtdm_Coarseness + 0.17*CT-wavelet-LLH_ngtdm_Busyness + 0.31*CT-wavelet-LLL_glcm_MCC + 0.11*CT-logarithm_ngtdm_Complexity-0.07*CT-square_glszm_SmallAreaEmphasis.9/L), Lym (1.5*109/L) Hb (148 g/L), and EBV-DNA .To build a robust prediction model, we enrolled Rad-score and several clinical parameters, including patients’ sex, age, T-stage, N stage, Rad-score (RS), systemic immune-inflammation index (SII), neutrophils (NE), lymphocytes (Lym), hemoglobin (Hb), and plasma Epstein–Barr virus DNA (EBV-DNA). Continuous variables were transformed into categorical variables to get meaningful information on the basis of the optimal cut-off value, which was determined using the maximum Youden’s index. Cut-off values were calculated using the ROC curve, and the specific cut-off values are shown below: age (44 years), RS (0.27), SII (321.75), NE (3.8*10P < 0.001) and SUVmax (P < 0.033) were independent indicators of prognosis. The holistic results of univariate and multivariate analyses are shown in Table In multivariate analysis of LRFS, the results showed that RS  in the training cohort and 0.70 (95% CI 0.64–0.76) in the validation cohort. The AUCs of the nomogram  were found to outperform the 8th edition of the T-staging system  in predicting 2- and 3-year LRFS , and a significant difference was also noted in the validation group . The abovementioned cut-off value was identified using the ROC. The log-rank test and Kaplan–Meier curve revealed a significant difference between high- and low-risk patients’ local failure risk in the training cohort . This was consistent with our observations. The C-index in our study was 0.71 (95% CI 0.68–0.74); however, we had a considerably larger cohort than Lv et al.’s study (726 vs. 140) for the construction of predictive model.Owing to the useful information it provides regarding tumor load and aggressiveness, 18F-FDG-PET/CT has been widely used to predict survival in patients with NPC. In a comparison of PET and CT radiomics for predicting local tumor control in HNSCC, Bogowicz et al. found PET to be more accurate than CT18. Given the widespread use of 18F-FDG-PET/CT, SUVmax has often been acknowledged as a useful parameter for estimating the prognosis of patients with NPC20. We incorporated SUVmax, EBV-DNA, and other clinical variables in this study. SUVmax was the only significant clinical factor according to the findings of the multivariable Cox regression analysis. Several studies have examined the significance of SUVmax as a predictor of treatment response or outcome in patients with NPC22. In a study by Xie et al., the cut-off value for SUVmax of the primary tumor was observed to be 8.0, which is roughly consistent with our study (7.4), and both studies reported it to be correlated with local recurrence23. SUVmax is used to determine the degree of malignancy on the basis of the rate of fluorodeoxyglucose breakdown in tumors. A higher SUVmax indicates that the tumor is metabolically more active and is likely to show faster proliferation and have lower differentiation potential. Thus, SUVmax showed a significant correlation with LRFS in the present study.As studies pursued the exploration of predictive factors of local recurrence, more and more potential factors were identified. According to Xiong et al.’s research, EBV-DNA was a promising predictor of survival in patients with locally advanced NPC24. Jiang et al. also reported that T-stage was not adequately effective in classifying patients into separate categories depending on the risk of local recurrence25. EBV-DNA was not enrolled into the nomogram owing to its lack of significance in univariate and multivariate analyses. We attribute this finding to the lack of stringent quality assurance measures implemented during clinical testing. Preiksaitis et al. showed that only 13/28 worldwide labs reported EBV-DNA levels within acceptable standards, demonstrating considerable interlaboratory variance. Variations in the perception of the detection threshold and the time of specimen collection after therapy restrict the use of plasma EBV-DNA detection in regular clinical applications26.Interestingly, the T-stage showed significance in univariate analysis but not in multivariate analysis. Although patients with T4 NPC may have a higher recurrence risk, the negative results of T-stage could be explained by collinearity in T-stage and SUVmax because both represented tumor burden. Lu et al. identified no correlation between T-stage and local recurrence24. Zhang et al. developed a radiomics nomogram incorporating age, sex, N-stage, and Hb as clinical variables27. Zhao et al. also constructed a nomogram for OS that incorporated the variables of age, N-stage, sex, EBV-DNA, T-stage, NLR, LMR, LAR, and PNI28. In the present study, a radiomics–clinical nomogram was developed. This nomogram could effectively estimate the 2- and 3-year LRFS risk and was valuable for categorizing patients into high- and low-risk subgroups. Risk classification and identifying the high-risk individuals who are more likely to develop local recurrence could be of significant value for early clinical detection and timely intervention. Nomograms, which have been widely used to represent predictive models in cancer prognosis, are incredibly useful tools for clinical decision-making and are extremely valuable to both clinicians and patients. Nomograms can also facilitate personalized treatment based on the total score of significant variables. The allocation of proportions for different elements was determined based on the correlation coefficient in a rational manner. Furthermore, nomograms maximize the translation of complex regression equations into graphs, and the outcomes of predictive models are easier for patients to comprehend30.Numerous studies have focused on integrating multiple significant clinical factors into prognostic models. In the study of Lu, the clinical factors included age, sex, T-stage, and plasma EBV-DNA levels31. Second, our model still has scope for further improvement. The predictive model may perform better if the present model is integrated with genomics, thus expanding its knowledge so that it does not rely solely on spatial information. In addition, our algorithm may have scope for further enhancement by deep learning techniques. Finally, clinic personnel were relatively unfamiliar with the radiomics model and statistical analysis algorithms and found them to be quite complex. Practical tools, such as applications, should be developed in the future.Our research had some limitations. First, this investigation was conducted in an endemic region and a single hospital without external validation. Owing to the retrospective design of the study and a single data source, multicenter studies having larger cohorts with external validation are required to verify our results and improve the reliability of our radiomics study, thus enhancing the clinical application of prognostic signaturesIn conclusion, we constructed a radiomics–clinical nomogram to predict local recurrence of NPC in patients by integrating radiomics features. Our model exhibited good prediction accuracy and independent discriminatory capacity in local recurrence-free survival. It may assist in personalized risk classification, facilitate individualized treatment strategies, and monitor clinical processes.


BP), Robusta Maculata (RM), BP x Sasso (BPxS), and RM x Sasso (RMxS). RNA was extracted from the jejunum and spleen to assess the mRNA expression of IL-2, IL-6, IL-10, IL-18, IL-1β, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The heterophil:lymphocyte (H/L) ratio and intestinal histomorphometric evaluation were also calculated. We found that compared to the conventional system, the rearing system significantly affected the jejunum expression of IL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in free-range system. A significant interaction between the rearing system and the genotype was also shown. More specifically, local breeds showed a significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 is constantly upregulated in local breeds within the free-range system compared to Ross hybrids. We also found significantly increased H/L and mortality rates in the latter, compared to the local breeds in the free-range reared system. The jejunum morphology also demonstrated a significantly higher villus height in BP and BPxS compared to the Ross hybrids. Overall, the results of our study confirm that the intense selection for growth in broiler chickens may have reduced their ability to react to the environmental stimuli related to free-range systems, resulting in a lower adaptability to a free-range environment, thus making them inappropriate for any farming system other than the conventional one. On the contrary, local chicken breeds are able to adapt and survive in the free-range system of rearing, and represent a genetic resource especially when adaptability to free-range conditions is required.The majority of poultry meat used to be sourced from intensively housed birds. However, consumer preference has since demanded poultry producers develop more sustainable farming systems. Although free-range farming is considered beneficial for animal welfare, it is not as easy to standardize as an intensive system, which makes the choice of bird genotype appear crucial for alternative systems. In this study, we aimed to evaluate the effect of conventional and free-range rearing systems on the immune status, stress parameters, intestinal morphology and mortality in commercial hybrids (Ross 308) and local poultry strains, Bionda Piemontese ( Commercial broiler chicken strains are the result of successful selection programs for rapid growth and body conformation . The maiFAO)  could have features or genes, for example, a higher immune response, capable of adapting to extensive rearing systems . In suchFAO) , local bFAO)  and shouFAO) .H/L) is influenced by stressors and can be used as a hematological indicator of the stress response in chickens , the stress parameters (H/L ratio) and the intestinal morphology and mortality on broilers and on local poultry strains.Chickens were reared according to EU Regulations 834/07, 889/2008, and the Italian directives on animal welfare for experimental and scientific purposes. The Bioethical Committee of the University of Turin  positively evaluated and approved the experimental protocol with the Protocol Number ID: 251833.BP), Robusta Maculata (RM), BP x Sasso (BPxS), RM x Sasso (RMxS), and commercial hybrid (Ross-308). The animals were vaccinated against infectious bronchitis virus, Marek's disease virus, Newcastle disease, Gumboro disease, and coccidiosis and they received food and water ad libitum. The chicks were reared for the first 20 d in the brood, divided into 5 pens, 1 for each genotype. Each pen was 1 m wide and 2 m long and was covered with wood shavings (20 cm deep) as litter and equipped with a waterproof floor and walls. It was environmentally controlled with temperature ranging from 32°C to 20°C and relative humidity (RH) from 70 to 65% RH, respectively. At 21 d until slaughter, the birds were randomly divided into 2 different rearing systems: conventional (33 kg of meat per m²) and free-range (21 kg of meat per m²). Three replicates (n = 10 cockerels/5 genotypes/2 housing systems/3 replicates) were made for each genotype and system.This trial was performed from March to July 2021 at the poultry farm of the Department of Veterinary Sciences of the University of Turin . A local hatchery purchased 300 male chickens of 5 different genotypes, Bionda Piemontese  and stored at −80°C until biomolecular analyses were conducted.In the conventional system, the lighting schedule throughout the trial was 16 h light, 8 h darkness. The environmental conditions in the poultry-house, that is, temperature and RH, were set according to the Ross guidelines . No lighA blood smear was prepared from a drop of blood without anticoagulant and placed on a single glass slide for each animal. The smears were stained using May-Grünwald and Giemsa stains. A 1:200 Natt–Herrick solution was used to treat the blood samples for the total red and white blood cell counts using an improved Neubauer hemocytometer. One hundred granular  and nongranular (lymphocytes and monocytes) leukocytes were counted on the slide and the H/L ratio was calculated .A Quick-RNA Miniprep Kit (Zymo-Research) was used according to the manufacturer's instructions to extract total RNA from 50 mg of ground spleen and jejunum tissue. RNA quantity was calculated by a NanoDrop 2000 Spectrophotometer  and the integrity of RNA was examined by electrophoresis in a denaturing 1% agarose gel. In order to avoid genomic DNA contamination, 1 µg of total RNA was treated with 1 U of DNase I amplification grade (Invitrogen) and the successful removal of the genomic DNA was confirmed by directly amplifying the RNA extracts without reverse transcription. DNase-treated RNA (500 ng) was reverse-transcribed into cDNA using a mixed priming strategy (Oligo dT + Random Hexamers) with a PrimeScript RT Reagent Kit (Takara) and incubated under the following conditions: 37°C for 15 min and 85°C for 5 s.IL-2, IL-6, IL-10, IL-18, IL-1β, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The expression ratio of the genes of interest was normalized according to the abundance of β-actin and GAPDH as reference genes to adjust for any unbalanced samples and corrected for coexpression in the qRT-PCR. The qRT-PCR reaction was carried out with 5 μL of a 10-fold diluted cDNA in a final volume of 20 μL using 10 μL of SsoFast EvaGreen Supermix (BioRad). Amplification was performed in a CFX96 Touch instrument  under the following thermal conditions: 98°C for 3 min, followed by 40 cycles of 98°C for 10 s and a different annealing temperature for each primer pair, as reported in NTC) were included in any run. The expression level was normalized using the ΔΔ Cq method (q method . FollowiVh) and crypt depth (Cd). In short, Vh was measured from the villus tip to the villus-crypt junction, whereas Cd was measured from this junction to the base of the crypt; the Vh/Cd ratio was also evaluated . Tissue slices were masked from the treatments and examined under a light microscope (Nikon Eclipse 800) equipped with a digital camera and images were acquired at 4× magnification. Morphometric evaluation was performed by the software Image J  measuring villus height  and slow-growing . Discriminant analysis (DA) was used to investigate differences between groups and to identify the variables that contribute most to differentiate these groups. The contribution of each variable to the discriminant function (Df) was expressed as structure coefficient, whereas the centroids, indicating the mean discriminant scores of Df for each group, were used to establish the cutting point for classifying samples. Eigenvalues were used to evaluate the relative discriminating power of each Df and the proportion of variance between groups was reported as a further measure of effect sizes. Lastly, Wilks’ lambda was used to test the significance of each Df. The performances of the final Df were estimated by classification statistics   were selected to satisfy the assumption of independence. Among them, variables to be included as predictors in the multivariate analysis were selected to balance the number of predictors and the sample size. Only variables with x table) .PCA). A principal component (PC) was extracted and items with loadings greater than |0.4| were interpreted. Cronbach's alpha (α) was used to estimate its reliability and values of >0.7 were considered acceptable (GLMs). Normal and Identity were set as the probability distribution and the link function, respectively. The effect of the rearing system , genotype (2 levels: slow- and fast-growing) and their interaction were evaluated, and the least significant difference was used for multiple comparisons. Multivariate analyses were conducted with SPSS Statistics version 25 . The level for statistical significance was set at P ≤ 0.05.The variables selected by univariate and discriminant analyses were then used to build a dimension describing the immune response of animals using a principal component analysis  and depended on the strain × rearing system interaction (P ≤ 0.05). We observed an increased H/L ratio in all the local breeds reared in a conventional system compared to the free-range birds and Ross strains showed a significant increased H/L ratio compared to the local breeds in controlled housing. The mortality rate was affected by genotype (P < 0.05), rearing system (P < 0.05), and their interaction (P < 0.05). Ross chicken mortality was significantly higher in both rearing systems, especially for free-range birds. The causes of Ross mortality were related to fast growth and the majority of deaths in the conventional system were due to sudden death syndrome (SDS) (65%) and ascites (35%), whereas mainly ascites (80%) was reported among the free-range birds. On the contrary, local breeds had a very low mortality rate (0.0–0.5%) in the free-range system.IL-2, which was significantly higher (P < 0.05) in the free-range system compared to the conventional one  when compared to fast-growing broilers.The rearing system did not affect the cytokine gene expression in the spleen, except for onal one . No signIL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in the free-range system compared to the conventional system. Significant interaction between the rearing system and the genotype was also displayed. More specifically, BPxS had a higher expression of IL-10 (P < 0.001) in the free-range system compared to the same genotype in a conventional system. BP, BPxS, and RM showed significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 was constantly upregulated in BP, BPxS, and RM compared to Ross in the free-range system. BP and BPxS showed a higher iNOS expression (P < 0.05) compared to the free-range reared Ross. No significant differences or interactions were observed in the expression of IL-18, IL-1β, IFN-γ, and TLR-4.IL-6, IL-2, and iNOS with positive loadings and H/L with a negative loading, whereas Df 2 included H/L and IL-10, both with positive loadings. The values of eigenvalue and Wilk's lambda indicated that Df 3 did not play a significant discriminatory role and, therefore, it was not interpreted and analyzed further. As regards Df1, the centroid  mainly discriminated the response of the 2 groups of genotypes reared free-range. In this system, slow-growing birds had positive scores , whereas fast-growing birds had negative scores . In Df2, the greatest difference between centroids was found between the fast free-range group (2.318) and the fast conventional group (−1.670). This suggests that Df2 (y-axis) mainly characterized the responses of the fast-growing broilers to the rearing system: the latter had positive scores  in the free-range system and negative scores in the conventional system.A multivariate analysis enabled the combined relation between the different traits to be considered. IL-2, IL-6, and iNOS with a positive loading and H/L with a negative loading. The low coefficient of IL-10 indicated the low contribution of this variable to the PC which, in turn, showed an acceptable, internal consistency (α > 0.7). The PC scores (P < 0.001), which interacted with the rearing system (P < 0.001). Fast-growing broilers had negative scores in conventional systems, which tended to further reduce when they were reared in the free-range system (P = 0.062). Negative PC scores indicated low values for IL-2, IL-6, and iNOS and high values for H/L. Slow-growing birds, on the other hand, had negative scores in the conventional system, although they significantly increased in the free-range system (P < 0.001). The increase in the PC scores indicated an increase in IL-2, IL-6, and iNOS values increased, whereas the H/L values decreased.Variables included in the first 2 Dfs were then selected for the PCA . One bipC scores  were infP < 0.001) in BP and BPxS reared in the free-range system compared to the same genotype in the conventional system. Moreover, within the free-range rearing system, BP and BPxS showed significantly higher values of Vh compared to Ross. All the local breeds reared in the conventional system showed a significantly lower Cd (P < 0.001) and higher Vh/Cd ratio (P < 0.01) compared to Ross.IL-2 has an important role in the immune response to antigenic stimuli; IL-1β and IL-6 are produced by monocytes and macrophages and act as proinflammatory cytokines with a relevant role in early phases of inflammation by eliminating pathogens from the host. IL-10 is a pivotal anti-inflammatory cytokine, able to inhibit proinflammatory cytokines. When proinflammatory cytokines are expressed in large quantities, the host can regulate the inflammatory response by upregulating the expression of anti-inflammatory cytokines. This results in positive feedback to maintain immunity balance . The resource allocation hypothesis is also consistent with our results on the H/L ratio of Ross chickens, which were higher than the results for the local breeds in both housing systems. Heterophils and lymphocytes are the 2 most abundant white blood cell types in birds, which play an essential role in innate and adaptative immunity, respectively , whereas the fast-growing birds had negative scores , which confirmed that local breeds are characterized by a certain degree of adaptability toward the environmental conditions. Several authors have conducted a comparative analysis between indigenous and exotic ovine  induction in chicken macrophages , the stress parameters (H/L ratio), and gut health  could be useful for finding strategies for the conservation and genetic improvement of local breeds. These breeds are in danger of extinction worldwide due to their replacement by more productive poultry strains. However, in addition to conserving biodiversity, they might represent a genetic resource of high-quality products, especially when adaptability to free-range conditions is required.In conclusion, our work suggests that both genotype and the rearing system play a role in the animal's immune response and welfare. Local breeds are able to cope with the extensive system better than the fast-growing, commercial strains, thus making the former an important genetic resource for alternative production systems. Adaptability to organic and free-range systems involves multiple parameters. Therefore, in addition to the daily weight gain, behavior, and welfare, other aspects related to immunity, stress, and gut health should be taken into account in order to establish a viable adaptability index.


A glioma is a malignant brain tumor with a poor prognosis. Attempts at the surgical removal of the tumor are the first approach, but additional treatment strategies, including radiation therapy and systemic or local chemotherapy, are necessary. Furthermore, the treatments are often associated with significant adverse side effects. Normal and malignant cells generally have antigenic differences, and this is the rationale for clinical immunotherapeutic strategies. Cytokines such as IL-15 or IL-2, which stimulate an anti-tumor immune response, have been shown to have a particularly high potential for use in immunotherapy against various tumors. In this review, treatments with either a poxvirus, genetically engineered to secrete IL-15, or allogeneic fibroblasts, transfected with tumor DNA and engineered to secrete IL-2, are shown to be effective strategies in extending the survival of mice with malignant brain tumors upon intracerebral injection of the treatment cells. Future studies with these treatment strategies in patients with intracerebral tumors are urgently needed. Some increase in the ability to diagnose and surgically treat primary brain tumors has been achieved, although the mortality and overall survival of patients with these tumors has not improved over many years . The pre+ cytotoxic T-lymphocyte (CTL) responses against the patient’s malignant cells [Normal and malignant cells generally have antigenic differences, which are the basis for clinical immunotherapeutic strategies. Several different strategies have been attempted to enhance anti-tumor immune responses in mice and patients with intracerebral neoplasms. Immunization with dendritic cells that have been “fed” derivatives of tumor cells or transfected with tumor RNA can result in the development of immune responses against the tumor antigens expressed by malignant cells ,7. In pant cells . Novel ant cells . Althougnt cells .Another strategy involves the preparation of a vaccine, prepared by the transfer of a cDNA expression library derived from tumor cells into an allogeneic mouse fibroblast cell line expressing a cytokine such as IL-2, which appears to have great potential for the development of an anti-tumor immune response, in the treatment of an intracerebral tumor, in mouse models . Upon thIn many aggressive tumors, such as gliomas, progression is enhanced by local immunosuppression, associated with an increase in regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) . The lacThere are several immunomodulatory cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which belong to the family of four α-helix bundle cytokines . The devA variety of tumor vaccination strategies have been attempted, including the modification of neoplastic cells to stimulate anti-tumor immune responses. Immunization with tumor cells, modified to secrete immune-augmenting cytokines such as IL-2, IFN-γ and GM-CSF, has resulted in the development of generalized MHC-restricted anti-tumor immune responses in animal models ,26,27,28Oncolytic virus is a type of virus, either engineered or in nature, which may selectively infect and lyse tumor cells while not affecting normal cells . There aOnce oncolytic viruses infect tumor cells, they may contribute to the anti-tumor response through a direct cytotoxic effect on tumor cells and the consequent release of tumor-associated antigens, which could stimulate anti-tumor immune responses, turning a “cold” tumor into a “hot” tumor . When thGM-CSF) was the first oncolytic virus approved for the treatment of advanced melanoma by the U.S. Food and Drug Administration (FDA) in 2015 [Many oncolytic viruses have already been tested in several preclinical and clinical trials. T-VEC  is a neuro-attenuated recombinant poliovirus (Sabin vaccine strains), whose internal ribosomal entry site (IRES) was replaced with human rhinovirus type 2 (HRV2) . The resPoxvirus, a group of large, enveloped DNA viruses associated with diseases that generate poxes in the skin, can also be a good choice for oncolytic virotherapy, since the entire replication of poxvirus happens in viral factories within the cytoplasm of infected cells, with no integration of viral DNA into the host genome, which is safe for host cells . PoxviruDespite the promising results, some concerns still need attention when using oncolytic viruses. Safety issues remain a major concern for oncolytic virotherapy. For example, vvDD vaccinia virus, which has undergone two phase I clinical trials and has been found to be safe in humans ,55, can b). LM fibroblasts are derived from C3H/He mice and express H-2k determinants. The potential development of an anti-tumor immune response was explored, using fibroblasts engineered to secrete either IL-2 or IL-2 and interferon-γ, injected into the brains of mice with an intracerebral (i.c.) glioma [p < 0.025). The results were more dramatic upon i.c. injection of mice with a glioma treated with LM-IL-2/interferon-γ double cytokine-secreting cells (p < 0.005). The i.c. injection of mice with equivalent numbers of LM-IL-2 cells without a tumor lived many months and did not demonstrate either ill effects or a neurologic deficit.Gl261 cells are a glioma cell-line of C57Bl/6 mouse origin (H-2) glioma . Glioma p < 0.05). This demonstrates that an anti-tumor immune response developed systemically in the mice with gliomas, following the intracerebral injection of cells secreting either IL-2 or IL-2/IFN-γ. Antibody depletion studies reveal that CD8+ and NK/LAK cells were responsible for the anti-tumor immunity.A chromium release assay was used to determine the reactivity of spleen cells from the immunized mice to chromium-labeled Gl261 glioma cells. The results  demonstr+ T cells). Rapamycin was used to enhance the spread and replication of oncolytic viruses [Two oncolytic poxviruses, the vvDD vaccina virus and the myxoma virus, were engineered to express the fusion protein IL15Ra-IL15 . Viral g viruses ,63,64, w viruses . The rat viruses . This su viruses .+ T cells was detected in the tumor specimens  indicated that myxoma virus (vMyx-IL15Rα-tdTr), supplemented by rapamycin and the prostaglandin inhibitor celecoxib, could provide a safe and effective anti-tumor treatment in mice with intracerebral gliomas, upon the injection of treatment cells into the brain or lateral ventricles. The efficacy of this novel combination treatment strategy in glioma-bearing mice, following tumor resection, is being explored [To explore the anti-tumor efficacy of myxoma virus expressing IL15, the combination treatment  was applied to mice with gliomas. An increased number of infiltrating NK and CD8pecimens A,B indicexplored .K) into cytokine-secreting mouse fibroblasts (H-2K). The application of these cells as a potential treatment of an intracerebral breast neoplasm arising in C3H mice was investigated. The cells were modified to express H-2Kb determinants, and this was intended to also ensure rejection. Studies with these cells revealed that there was a prolongation of survival in C3H mice with intracerebral breast cancer, upon treatment by immunization with fibroblasts secreting either IL-2 or GM-CSF transfected with DNA from the same spontaneous breast neoplasm (p < 0.05) [A tumor vaccine was constructed by the transfer of DNA from a breast cancer cell line (SB-5b) that arose in C3H mice (H-2 < 0.05) .An ELISPOT-IFN-γ assay was used to estimate the development of splenic T cells, reactive with SB-5b cells, in mice immunized with transfected fibroblasts modified to secrete IL-2 or GM-CSF. The assay was performed six weeks after the i.c. injection of the mixture of SB-5b cells and the transfected fibroblasts. The findings in these studies revealed that the highest proportion of T cells reactive with SB-5b cells was in surviving mice injected with fibroblasts modified to secrete IL-2. Lesser numbers of spots were found in T cells from mice injected with SB-5b cells and non-secreting transfected fibroblasts or SB-5b cells and transfected fibroblasts modified to secrete GM-CSF.bIL-2/SB5b cells. An ELISPOT assay was undertaken after two weeks, using the spleen cells to detect IFN-γ secretion in the presence or absence of SB-5b tumor cells and antibodies against various T cell subsets. These studies revealed that CD4+, CD8+ and NK/LAK cells were responsible for the anti-tumor immune response. The overall p-value between the unstimulated and the tumor cell stimulated group was p < 0.001.In additional experiments, animals with i.c. breast cancer were treated i.c. with LMKhigh pool) and least (immunolow pool) after three rounds of enrichment were identified and used in further studies.A strategy was developed to enrich the vaccine for efficacy by identifying cell populations that were the most highly immunogenic . Populat6 cells from the immunohigh pool on two days separated by one week. For the controls, the same procedure was followed, except that the cells from the non-enriched master pool or cells from the immunolow pool were substituted for cells from the immunohigh pool. For the additional controls, the tumor-bearing mice were injected into the brain with equivalent numbers of non-DNA-transfected LMKb cells or with SB5b tumor cells alone. Mice injected with SB5b tumor cells received only one injection. The data revealed that the highest number of responding T cells were in the cervical lymph nodes (high pool (p < 0.05).To analyze the development of systemic anti-tumor immunity in tumor-free mice injected i.c. with cells from the various treatment groups, ELISPOT IFN-γ assays for the measurement of responding T cells were carried out using cells from the cervical lymph nodes and spleens from the injected mice. A micro cannula was placed into the right frontal lobe of naïve C3H/He mice. Following the cannula insertion, the animals were injected into the brain through the cannula with 1.0 × 10ph nodes A or spleph nodes B of micehigh pool, using the above protocol. ELISPOT IFN-γ assays were used for this analysis. The anti-tumor immune response was inhibited to the greatest extent by antibodies against CD4+ cells. The results were less dramatic if the spleen cells were incubated in the media containing CD8+ or NK/LAK antibodies.The effect of antibodies against various T cell subsets on the responding T cell response was used to determine the types of cells activated for anti-tumor immunity in the spleens of mice treated with cells from the immunohigh pool. The findings from these studies reveal that Foxp3+ T-reg cells were relatively deficient in the spleens from the animals with i.c. breast cancer treated with cells the immunohigh pool. An analysis of the spleen cells by FACS revealed that mice injected i.c. with cells from the immunohigh pool had a relative deficiency of CD4+/CD25+/Foxp3+ T cells, and a corresponding increase in the numbers of CD8+ cells.T-reg cells are potent inhibitors of natural anti-tumor immunity . Protocohigh pool, C3H/He mice were injected i.c. with 5.0 × 104 SB5b cells and 1.0 × 106 cells from the immunohigh pool. The findings from these studies revealed that mice with i.c. breast cancer treated with cells from the immunohigh pool survived significantly longer than untreated mice (p < 0.05); the treated animals survived 26 days while the untreated animals survived only 14 days. Similar results were demonstrated if the mice with i.c. breast cancer cells were treated with the subcutaneous (s.c.) injection of cells from the immunohigh pool.To explore the potential prolongation of survival in mice with i.c. breast cancer treated by cells from the immunoThe heterogenicity and weakly immunogenic composition of gliomas makes these tumors difficult to treat. A limited number of phase III immunotherapy trials have been completed for checkpoint inhibitors, monoclonal antibodies and dendritic cell vaccines, which have not demonstrated an improvement in survival outcomes ,70. One In summary, the data from this study suggest that a poxvirus genetically engineered to secrete IL-15, along with a prostaglandin synthesis inhibitor to block immunosuppression, is an effective treatment for increasing survival in mice with a malignant glioma. Two different oncolytic poxviruses expressing the IL15Rα-IL15 fusion protein were studied. Cytokines such as IL-2 and IL-15 are beginning to find a role in the management of patients with malignant gliomas ,75. Earl+ T cells [Oncolytic viruses may contribute to the anti-tumor response through direct cytotoxic effects on cancer cells, such that there is a release of tumor antigens which may stimulate an immune response. Previous studies have shown that the oncolytic poxvirus, myxoma virus, is safe to use in mice even when directly injected into the cerebral ventricles . When th T cells  with les T cells  and less T cells .In other studies, it was shown that a vaccine prepared by the transfer of a cDNA expression library, derived from tumor cells, into a mouse fibroblast cell line engineered to secrete IL-2 appears to be efficacious in the treatment of a brain tumor. The cDNA integrates spontaneously into the genome of the recipient cells, which is followed by replication and expression. The vaccine requires only a small amount of tumor tissue for preparation and can be given at an early stage of the disease, when the tumor is most susceptible to immune-based therapy. A unique enrichment strategy was also developed, such that the proportion of active immunotherapeutic cells in the vaccine is increased.high pool were CD4+ T cells. The treatment of a brain tumor with cells from the immunohigh pool also resulted in a reduction in the relative numbers of T-reg cells, with a potential prevention of the impaired anti-tumor immune responses frequently found in patients with malignant brain tumors.The use of cells from the enriched vaccine was associated with the development of a strong anti-tumor immune response. The predominant cell type activated in mice immunized with cells from the immunoThe goal of tumor treatment would be the removal of every tumor cell. A single therapy will likely not achieve this goal in the case of brain tumors. However, immunotherapy, in combination with surgical removal of the tumor, radiation therapy and chemotherapy, will likely be involved in the management of patients with brain tumors. The development of DNA-based tumor vaccines, in combination with cytokine secretion, is a novel strategy that does not require antigen identification or protein purification, and yet can elicit a potent and long-lasting activation of the immune response, which will lead to the rejection of the tumor. Only small quantities of tumor-derived DNA, from surgical specimens, are needed to prepare the vaccine. The enrichment strategy represents a unique approach to isolating highly immunogenic pools of transfected cells, which leads to the development of enhanced anti-tumor immunity. Thus DNA-based cytokine-secreting vaccines offer several unique advantages, which support their further development as an immunotherapeutic treatment option for patients with a variety of tumors in general and, in particular, patients with malignant intracerebral tumors. The use of viruses engineered to secrete cytokines remains another treatment option and requires further study regarding the management of these tumors.


The aim of this study was to investigate the association between inflammatory plasma protein concentrations and long-term mortality in patients with ST-elevation myocardial infarction (STEMI).For 343 STEMI patients recorded between 2009 and 2013 by the population-based Myocardial Infarction Registry Augsburg, 92 inflammatory plasma proteins were measured at the index event using the OLINK inflammation panel. In multivariable-adjusted Cox regression models, the association between each plasma protein and all-cause long-term mortality was investigated. Median follow-up time was 7.6 (IQR: 2.4) years. For plasma protein that showed a strong association with long-term mortality, a 5-year survival ROC analysis was performed.p-value of <0.001 and a Hazard Ratio (HR) of 1.57 [95% CI: 1.29–1.91]. In the 5-years ROC analysis, an AUC of 0.6903 [95% CI: 0.594–0.781] was estimated for FGF-23. All other plasma protein didńt show strong associations, each marker with FDR-adjusted p-values >0.05 in the multivariable-adjusted Cox models.One plasma protein, namely Fibroblast Growth Factor 23 (FGF-23), was particularly well associated with long-term mortality in the multivariable-adjusted Cox model with an FDR-adjusted FGF-23 is independently associated with long-term mortality after STEMI and might play an important role in the response to myocardial injury. The results suggest FGF-23 to be a useful marker in the long-term treatment of STEMI patients and a potential target for drug development. Never2.2.1.This study is based on data from the population-based Augsburg Myocardial Infarction Registry. It was established in 1984 as a part of the MONICA-project (Monitoring Trends and Determinants in Cardiovascular disease) and operated as KORA Myocardial Infarction Registry  until 202.1.1.Trained study nurses interviewed the participants during hospital stay using a standardized questionnaire. In order to confirm the information provided by the patients and to collect additional information, the patients' medical chart was reviewed. Demographic data, data on cardiovascular risk factors, medical history, comorbidities (including diabetes) and medication before and during hospital stay, as well as at discharge were collected from each patient. Furthermore, routine laboratory parameters including glucose measurement, ECG results and data on the in-hospital course were assessed.Between 2009 and 2014 arterial EDTA blood samples were obtained immediately after hospital admission, right at the beginning of the cardiac catheterization. After centrifugation and aliqoting in the catheter laboratory, EDTA plasma samples were stored frozen at −80°C until laboratory analysis.2.1.2.Measurements of the 92 proteins were performed in plasma samples using the Proseek® Multiplex Inflammation panel, developed by Olink Proteomics  and based on the Proximity Extension Assay (PEA) technology. Detailed information on the process of measurement can be found in a previous publication .All other blood parameters were measured in clinical laboratory at the university hospital of Augsburg during hospital stay of the patients as part of the regular diagnosis and routine treatment.2.1.3.The endpoint used in this study was long-term all-cause mortality. Mortality was ascertained by regularly checking the vital status of all registered persons of the MI registry with data from the population registries. Death certificates were obtained from local health departments. The present analysis included patients with ST elevation myocardial infarction (STEMI) only. All patients who died within the first 28 days after AMI were excluded in order to concentrate on long-term mortality exclusively. Overall, 343 STEMI patients survived the first 28 days after infarction and were considered for this analysis.2.2.χ2 tests were performed and the results were presented as absolute frequencies with percentages. For normally-distributed continuous variables, Student's t-tests were used. For continuous variables that were not normally-distributed we used nonparametric tests. The results were presented as mean and standard deviation (SD) or median and inter-quartiles range (IQR).For the comparison of categorical variables, 2.2.1.p-values were false discovery rate (FDR)-adjusted. In a subsequent step, the same Cox regression models were calculated but potential confounder variables were added according to a literature review. These multivariable-adjusted Cox regression models were adjusted for sex, age, glomerular filtration rate , diabetes mellitus, hypertension, hyperlipidemia and percutaneous coronary intervention (PCI), yes/no).First, the obtained values for each plasma protein were standardized in order to ensure comparability between the different markers. To examine the associations between the 92 plasma proteins and long-term mortality, 92 Cox regression models were calculated, one for each protein. The initial models were adjusted for sex and age. To control the effect of multiple testing, the obtained The proportional hazards assumption was checked by plotting the Schoenfeld residuals against time and searching for any visible correlation and by testing for a significant correlation of the Schoenfeld residuals with time. The proportional hazards assumption was considered to be sufficiently fulfilled for all models.As a sensitivity analysis, two more multivariable adjusted Cox regression models were calculated, each including the same covariables as the main model but adding either prehospital time or left ventricular ejection fraction (LVEF) see , S2. Fin2.2.2.For plasma proteins that were strongly associated with long-term mortality ROC analyses was performed to quantify their predictive ability.Best cut-off value for a dichotomization of the plasma protein markers were determined by calculating Youden-Indices. The sample then was divided in a “high plasma protein” group and a “low plasma protein” group according to the maximized Youden-Index. Finally, a Kaplan-Meier curve was plotted and stratified by the dichotomous variable high/low plasma protein group.3.For a total of 343 STEMI cases data of 92 inflammation-related plasma proteins were available for the statistical analysis. p-value of <0.001 and a Hazard Ratio (HR) of 1.57 [95% CI: 1.29–1.91] per SD.As for most plasma proteins 3 values were missing, a total of 340 cases was included into the Cox regression models . Results of the Cox regression models adjusted for sex and age are displayed in In the subsequently performed 5-years ROC analysis, 339 patients were included . The number of events (=death within 5 years) was 44 and the number of controls was 295. 4.In this study, we investigated the association of 92 plasma proteins with long-term mortality in ST-elevation myocardial infarction patients. One protein, FGF-23, exhibited a particularly strong association with long-term mortality in the multivariable-adjusted Cox regression model  as well as in a 5 years ROC analysis . All other plasma proteins were not significantly related to long-term mortality in the multivariable-adjusted Cox regression model.FGF-23 is a representative of the fibroblast growth factor family and is classified by OLINK as a cancer marker. It is mainly produced by osteoblasts and osteocytes but can also be produced and secreted by other tissue cells . Several4.1.In a meta-analysis on FGF-23 and risks of cardiovascular and non-cardiovascular diseases the authors concluded that the association between FGF-23 and the diseases might be non-causal . In a ra4.2.It has been reported that high plasma FGF-23 concentrations are independently associated with LV remodeling after STEMI . Patholo4.3.2), so we conclude that the association between FGF-23 and mortality after AMI is (mainly) independent of renal function or CKD.Other pathophysiological pathways might potentially be involved in the association between plasma FGF-23 and long-term outcome after STEMI. First, FGF-23 is known to be strongly connected to CKD, which is a major risk factor for adverse outcomes in AMI patients –29. Cons4.4.The scientific literature moreover suggests a relation between FGF-23 and heart failure/reduced left-ventricular ejection fraction –32. FGF-4.5.The blood samples in this study were very uniformly taken at the beginning of the cardiac catheterization. As we included only STEMI events, the delay between hospital admission and PCI was generally very small. Nevertheless, greater deviations between the patients were observed in prehospital time, which represent the majority of therapeutic delay and negatively affects outcome in AMI patients , 39. Tak4.6.This study is characterized by some strengths. First, it is based on patients from the population-based Augsburg Myocardial Infarction Registry with consecutive enrollment of patients, which minimizes the effect of selection bias. Blood samples were uniformly taken during the PTCA intervention, guaranteeing highest consistency in blood sampling. Moreover, for every case there was a large number of additional information which we used for proper adjustment in the Cox regression models.Nevertheless, there are also some limitations. We have no validation cohort from another register for our analyses, which means we cannot validate the associations found in this study. Moreover, as this study is based solely on observational data, we cannot draw any conclusions about causality . Additionally, we might not have considered all potential confounders. Last, as this study included only patients between 25 and 84 years with STEMI, the results may not be generalized to all age or ethnic groups as well as to Non-ST-elevation MI events.5.Elevated FGF-23 plasma concentrations are independently associated with higher long-term mortality in STEMI patients. The effects appear to remain stable over the course of one decade. FGF-23-concentrations at baseline might help to identify a population in the need of intensive and lasting secondary prevention measures to target long-term mortality. Moreover, if a causal nature of the relationship can be established, new therapies targeting FGF-23 might represent a promising approach especially in the long-term treatment of patients after AMI.